Many enzymes have inefficient promiscuous activities. Promiscuous activities can be patched together to facilitate low-flux pathways that we have termed serendipitous pathways. Serendipitous pathways become important when a mutation raises the level of flux or when the environment changes such that even low flux improves fitness or survival. This proposal addresses the evolution of new metabolic pathways that have been patched together from promiscuous enzyme activities. This work has important implications for molecular evolution, both in the past, and in the present in response to selective pressures exerted by anthropogenic compounds such as pesticides, munitions and chemotherapeutics. Pathways for degradation of pesticides such as atrazine and pentachlorophenol likely arose from serendipitous pathways that provided a novel source of nitrogen or carbon or eliminated a toxin. Further, serendipitous pathways might foster resistance to anti-metabolite drugs in situations involving very large populations (e.g. pathogenic bacteria) or high mutation rates (e.g. tumors). We will explore how the resources within a proteome can be utilized to assemble serendipitous pathways and how cells can adapt to use such pathways more efficiently in the context of a model system in E. coli. We have discovered that E. coli has at least three serendipitous pathways that allow synthesis of the essential cofactor pyridoxal phosphate when an enzyme in the normal biosynthetic pathway is missing. One of seven different genes must be over expressed to increase flux through these serendipitous pathways to a level that supports growth. We have characterized one of these pathways. Aim 1 describes efforts to characterize a second pathway to further characterize how the thousands of promiscuous activities available within a cell can be patched together to provide a novel pathway. The serendipitous pathways we have discovered are inefficient. Furthermore, the pathway we have characterized uses two unusual metabolites that are toxic. We have resequenced the genomes of 18 strains of E. coli that have been adapted to grow more efficiently on glucose while using serendipitous pathways to supply PLP. Aim 2 describes plans to characterize the molecular mechanisms of the mutations that allow E. coli to use a serendipitous pathway more effectively. Different microbes contain different complements of enzymes. Furthermore, the levels of promiscuous activities between orthologs in different microbes can vary significantly. Thus, the potential for combining promiscuous activities into serendipitous pathways should vary among microbes. Aim 3 will examine how the resources available in the metabolic network of Salmonella enterica can be used to meet the challenge of synthesizing PLP when the normal pathway is disabled.